Multiplicity-edited pulse sequence

Determination of the number of directly bound hydrogen atoms, called multiplicity assignment, is easy through application of editing pulse sequences, e.g., distortionless enhancement by polarization transfer (DEPT) or attached proton test (APT). 

The HSQC experiment is based on single rather than multiple quantum coherence during the evolution time, t. The contemporary multiplicity-edited gradient HSQC pulse sequence is shown in Fig. 10.15. Relative to the much simpler HMQC pulse sequence, the HSQC... 

Fig. 10.15. Pulse sequence for the multiplicity-edited gradient HSQC experiment. Heteronuclear single quantum coherence is created by the first INEPT step within the pulse sequence, followed by the evolution period, t . Following evolution, the heteronuclear single quantum coherence is reconverted to observable proton magnetization by the reverse INEPT step. The simultaneous 180° XH and 13C pulses flanked by the delays, A = l/2( 1 edits magnetization inverting signals for methylene resonances, while leaving methine and methyl signals with positive phase (Fig. 16A). Eliminating this pulse sequence element affords a heteronuclear shift correlation experiment in which all resonances have the same phase (Fig. 16B).

Using strychnine (1) as a model compound, a pair of HSQC spectra are shown in Fig. 10.16. The top panel shows the HSQC spectrum of strychnine without multiplicity editing. All resonances have positive phase. The pulse sequence used is that shown in Fig. 10.15 with the pulse sequence operator enclosed in the box eliminated. In contrast, the multiplicity-edited variant of the experiment is shown in the bottom panel. The pulse sequence operator is comprised of a pair of 180° pulses simultaneously applied to both H and 13C. These pulses are flanked by the delays, A = l/2(xJcii), which invert the magnetization for the methylene signals (red contours in Fig. 10.16B), while leaving methine and methyl resonances (positive phase, black contours) unaffected. Other less commonly used direct heteronuclear shift correlation experiments have been described in the literature [47]. 

Fig. 10.16. (A) GHSQC spectrum of strychnine (1) using the pulse sequence shown in Fig. 10.15 without multiplicity editing. (B) Multiplicity-edited GHSQC spectrum of strychinine showing methylene resonances (red contours) inverted with methine resonances (black contours) with positive phase. (Strychnine has no methyl resonances.) Multiplicity-editing does have some cost in sensitivity, estimated to be 20% by the authors. For this reason, when severely sample limited, it is preferable to record an HSQC spectrum without multiplicity editing. Likewise, there is a sensitivity cost associated with the use of gradient based pulse sequences. For extremely small quantities of sample, non-gradient experiments are preferable.

The INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) experiment [6, 7] was the first broadband pulsed experiment for polarization transfer between heteronuclei, and has been extensively used for sensitivity enhancement and for spectral editing. For spectral editing purposes in carbon-13 NMR, more recent experiments such as DEPT, SEMUT [8] and their various enhancements [9] are usually preferable, but because of its brevity and simplicity INEPT remains the method of choice for many applications in sensitivity enhancement, and as a building block in complex pulse sequences with multiple polarization transfer steps. The potential utility of INEPT in inverse mode experiments, in which polarization is transferred from a low magnetogyric ratio nucleus to protons, was recognized quite early [10]. The principal advantage of polarization transfer over methods such as heteronuclear spin echo difference spectroscopy is the scope it offers for presaturation of the unwanted proton signals, which allows clean spec-... 

The development of carbon-13 NMR during the last eight years has been characterized by a continual increase in the sensitivity and quality of spectra. A reduction in measuring time - equivalent to an enhancement in sensitivity has been achieved mainly by cryomagnet technology. The efficiency with which NMR information can be obtained has been substantially improved by new computer-controllable pulse sequences for one-and two-dimensional NMR experiments. A selection of these new methods, in particular, those used for multiplicity analysis and homo- or heteronuclear shift correlations, is presented in chapter 2 of this edition. 

The major breakthroughs, however, have come from the use of high magnetic fields and further from the use of different multiple pulse sequences to manipulate the nuclear spins in order to generate more and more information time domain NMR spectroscopy, that is used to probe molecular dynamics in solutions. The latter made it also possible to "edit" sub-spectra and to develop different two-dimensional (2D) techniques, where correlation between different NMR parameters can be made in the experiment (e.g. SH versus 813c, see later). Solid state NMR spectroscopy is used to determine the molecular structure of solids. 

A simple modification of 2D 1,1-ADEQUATE and chemical shift ojj-refocused 2D 1,1-ADEQUATE has been proposed recently 38 An insertion of a 180° aH pulse into the initial 13C-13C spin-echo yields cross peaks edited by the CHV multiplicity the cross peaks of CH and CH3 groups display opposite phase to those of CH2 groups leading to distinct phase patterns facilitating spectral analysis. Although the length of the pulse sequence is not affected by this modification, losses of sensitivity can occur due to a mismatch between the set and actual value of [fc i coupling constants. 

An alternative pulse sequence that provides the same multiplicities as INEPT but with intensity ratios that follow the binomial theorem is DEPT (distortionless enhancement by polarization transfer).The pulse sequence, depicted in Fig. 12.3a, can be used, like refocused INEPT, for sensitivity enhancement but is usually employed as an editing technique. The three evolution periods T are chosen to approximate 1/2/, but the length of the pulse labeled 0 can be varied. As we show in the following, CH has maximum intensity at 0 = 90° CH2 has zero... 

A short remark about the relevance of HMQC and HMBC experiments and nomenclatures should be done here. Initially, Bax et al. [9] introduced the HMQC technique for specific editing of H- C pairs correlated by direct V( C, H) couplings. The HMBC technique was proposed subsequently by Bax and Summers [11] to edit specifically multiple bond correlations through "/( C, H) couplings (n = 2 and 3), which explains the HMBC acronym. From the point of view of the pulse sequence the introduction of the low-pass filter, which consists of a supplementary 90° pulse and an extended phase cycle, is... 

Figure 7-25 The multiplicity-edited gradient HSQC pulse sequence. The relative strengths of gradients G and G2 are 4 and 1 G cm respectively as shown, when X = C.

Multiplicity editing in 2D NMR is possible via INEPT- or DEPT-HMQC [121] experiments. Choosing the editing pulse or delay, as in ID experiments, can yield spectra with CH and CH3 contours positive and CH2 contours negative [2,74]. Useful and potentially cleaner spectra can be obtained this way however, these sequences have been little utilized in lignin work to date. They are experiencing renewed interest with the adoption of the latest shaped-pulse variants. 

The processes that occur during the evolution period are probably the most important in describing the effect of the complete pulse sequence. During this period coherence can evolve, coherence can be selectively manipulated or coherence transfer can occur. Coherence manipulation can be the inversion of the coherence order (WATERGATE experiment) or in a l S spin system a phase shift depending upon signal multiplicity (APT or SEMUT experiment). In the case of heteronuclear IS spin systems the creation of antiphase coherence and subsequent polarization transfer using a INEPT or a DEPT unit can be used in multiplicity edited experiments or heteronuclear 2D correlation experiments. In transient NOE experiments such as ROE and TROESY, coherence... 

Looking at this second edition in a little more detail chapter 5.3.1 has been extended to include simulations of multiple offset selective pulse experiments whilst chapter 5.5.2 examines ACCORDION-principle based HMBC experiments. Multiple offset selective pulse experiments are now an important application in LC-NMR and biomolecular NMR spectroscopy whilst the discussion of the CIGAR and the IMPEACH-HMBC experiment in chapter 5.5.2 are an invitation to use NMR-SIM to trace complex pulse sequences back to their origin. There is now a more comprehensive discussion of filter elements, which are now a vital element in the latest pulse sequences, in chapter 5.8.2. Finally the new chapter 5.9 is subdivided into two subsections. The first subsection 5.9.1 is a collection of some of the latest published ideas to improve existing sequences. Section... 

Fig. 8.2 Pulse sequence for the DEPT experiment [41, 42]. By adjusting the variable flip angle read pulse, 6, it is possible to generate edited subspectra based on resonance multiplicity (CH, CH2, and CH3). When d = 45°, all protonated carbons will exhibit positive intensity. When 6 = 90°, only methine carbons are observed and have positive intensity. When 6 = 135° a spectrum is produced in which methine and methyl resonances have positive intensity...

Fig. 8.21 A. GHSQC spectrum of the aliphatic region of the strychnine (2) spectrum recorded using the pulse sequence shown in B. multiplicity-edited GHSQC [120-123] spectrum of strychnine showing methylene resonances in red and opposite in phase from...

Fig. 8.22 Schematic representation of the plicity editing step following the evolution multiplicity edited GHSQC pulse sequence in period [120-123]. use in the author s laboratory with the multi-...

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